The major challenge facing TBV development is to find a highly safe formulation that induces sustained high antibody responses. LMIV has demonstrated that conjugating Pfs25 and Pfs230 with carrier protein ExoProtein A (EPA) of Pseudomonas aeruginosa greatly enhances the immunogenicity of the recombinant TBVs and has shown to be safe with the adjuvant Alhydrogel, but may need a stronger adjuvant such as AS01 to achieve the antibody responses needed to block transmission. Highlighted in this years summary are results from our publications in FY19: 1. Reiter K, et al. Host Cell Protein Quantification of an Optimized Purification Method by Mass Spectrometry. 2019. Journal of Pharmaceutical and Biomedical Analysis. 2019. 174:650-654. Recombinant ExoProtein A (EPA), a detoxified form of Pseudomonas aeruginosa Exotoxin A, is used as a protein carrier for LMIV vaccine candidates. A scaled manufacturing process, in which EPA was expressed in Escherichia coli, yielded a product that approached or exceeded our upper limit of E. coli host cell protein (HCP) content per human dose. The purification process was redeveloped to reduce HCP levels in the bulk product and HCP content was evaluated by orthogonal methods. These improved HCP profiles support implementing the revised purification process for manufacturing the EPA protein carrier and 1D/LC-MS/MS for HCP analysis. 2. Coelho CH, et al. Transmission-blocking vaccines for malaria: Time to talk about vaccine introduction. 2019. Trends in Parasitology. Jul;35(7):483-486. Transmission-blocking vaccines (TBVs) follow a novel strategy by targeting the mosquito stages of the parasites life cycle. Given the recent progress inTBV product and clinical development, increased attention is needed to prepare for the introduction of TBVs in malaria-affected areas, particularly low-resource settings. Here, we outlined key discussions between communities, researchers, and country stakeholders that must occur in anticipation of introduction of TBVs in endemic, low-resource communities. In addition to these publications, we have seen progress in our trials of Pfs25 and Pfs230 vaccines: Pfs230D1M-EPA/AS01 and Pfs25M-EPA/AS01 in Healthy Malian Adults (NIAID protocol 17-I-N006). The phase 1 study of the safety, tolerability, immunogenicity, and functional activity of Pfs25M-EPA/AS01 (Pfs25M) and Pfs230D1M-EPA/AS01 (Pfs230) started recruitment for the Pilot Safety Phase dose-escalation in Sotuba in Dec 2016. Vaccinations were scheduled at All 3 vaccinations (0, 1, and 6 months) for all Arms enrolled in the Safety Pilot Phase of the study were completed. Results from the Pilot Safety Phase showed that Pfs230D1M-EPA/AS01 (Pfs230) alone exhibited higher antibody titers and functional activity that did not improve with additional of Pfs25M-EPA/AS01 (Pfs25M). As a result, the study continued with only administration of Pfs230D1M-EPA/AS01. Recruitment for the randomized double-blind portion of the study (Main Phase) started in Feb 2017, with pre-emptive treatment with Artemether Lumefantrine, en bloc randomization with stratification by village., and 2 of 3 vaccinations being completed in the 0, 1, 6-month vaccination schedule to date. The third and final vaccination was completed in Aug/Sept 2017 and beginning in Sep 2017 subjects in the Main Phase underwent 12 weeks of DSF (twice a week) for a total of 24 mosquito feeds. In August 2018, 163 participants re-enrolled and were vaccinated in Bancoumana and Doneguebougou villages, with 16 weeks of DSF assays planned to be completed in December 2018. The results to date in this study indicate that antibody titers are increased substantially with Pfs230D1M-EPA/AS01 and Pfs25M-EPA/AS01 over those achieved with Alyhdrogel-adjuvanted vaccines., In addition, that the functional activity induced by Pfs230D1M-EPA/AS01 is superior to Pfs25M-EPA/AS01 and is not improved by the addition of Pfs25M-EPA/AS01, and that significant functional activity can be achieved after only 2 doses of Pfs230D1M-EPA/AS01. The study follow up has continued in FY19, with the Last Subject Last Visit anticipated before the end of FY19. Safety, Immunogenicity and Efficacy of Pfs230D1M-EPA/AS01 Vaccine, a Transmission Blocking Vaccine against Plasmodium falciparum, in an Age De-Escalation Trial of Children and a Family Compound Trial in Mali (NIAID protocol 19-I-N086). The phase 2 study of the safety, immunogenicity, vaccine activity, and vaccine efficacy of Pfs230D1M-EPA/AS01 against Plasmodium falciparum malaria in family compounds in Doneguebougou, Mali started its Pilot Phase in May 2019 with initial enrollment into the Safety Arms 1A/1B (9-18 year olds) in April 2019 and then Arms 2A/2B (5-8 years olds) in May 2019. For these initial safety arms, 30 subjects aged 9-18 years of age were consented, screened, enrolled, and vaccinated (Vax #1: April 27/28, 2019; Vax #2: May 25, 2019) and 30 subjects aged 5-8 years of age were consented, screened, enrolled, and vaccinated (Vax #1: May 15/16, 2019; Vax #2: June 12, 2019). As Pilot Phase subjects underwent vaccinations and post vaccination follow-up, entire vaccine units (VU) compised of families or groups of familieis were approached for consenting, screening, enrollment, and vaccination. Per protocol, 30 VUs were initially enrolled and vaccinated: 5 VUs vaccinated on June 15, 2019, 10 VUs vaccinated on June 18, 2019, and the final 15 VUs on June 20 and 27, 2019. In total, an additional 25 5-8 years old, 103 9-18 years old, and 133 19 years old subjects were enrolled from these 30 VUs (n=261 in total). Subsequent to DSMB review of these 30 VUs, additional VUs continue to be enrolled and vaccinated with an enrollment goal of 1500 total vaccinated subjects. In FY19, study participants in the vaccination groups will receive 3 doses of Pfs230D1M-EPA/AS01 or comparator vaccine. During followup in the malaria season that started in FY19, vaccine activity will be measured in 9-18 yo children by mosquito direct skin feeding (DSF) assays to measure parasite transmission to mosquitoes, and vaccine efficacy will be measured in 1-8 yo children by blood smear diagnosis of new infections following clearance of parasitemia with antimalarial artemether-lumefantrine treatment at the onset of the malaria season.